The proposed research addresses potential molecular mechanisms underlying the formation of precise synaptic connections in the auditory brainstem. The applicant has previously shown that several members of the Eph family of receptor tyrosine kinases are expressed in avian auditory brainstem nuclei and along the midline during the formation of synaptic connections. These receptors mediate cell-cell interactions during axon outgrowth, and gradients of Eph receptors and their ligands, the ephrins, are required for the formation of precise topographic maps in the visual system. The Specific Aims are to test the roles of Eph receptor signaling in the development of auditory brainstem projections. Each group of experiments includes both descriptive studies of developmental expression and functional studies using experimental manipulations. Inovo microelectroporation will be used to misexpress Eph receptors and an in vitro labeling method will be used to evaluate resulting axonal projections. The generality of findings from chicks will be examined in developing mouse brainstem. Specific Aim 1 proposes experiments to investigate whether gradients of Eph receptors and ephrins are essential for the development of the tonotopic projection from VllIth nerve axons to the avian cochlear nucleus, n. magnocellularis. Specific Aim 2 extends these studies to mammals. The Eph receptor EphA4 is expressed nonuniformly in the developing mouse cochlear nuclei. The role of EphA4 in the establishment of a homologous projection from the VIIIth nerve to the cochlear nucleus will be evaluated in mutant mice that lack EphA4. Axonal projections from n. magnocellularis branch so that an ipsilateral branch synapses in the dorsal region of the target, n. laminaris, while the contralateral branch grows across the dorsal midline and contacts the ventral region of contralateral n. laminaris. Experiments outlined in Specific Aim 3 will evaluate the role of Eph receptors in the guidance of cochlear nucleus axons at the midline in the avian brainstem. These experiments will test whether Eph signaling is extensively used in the development of pathways in the auditory brainstem, and will address whether the roles for Eph receptors in establishing synaptic specificity are similar across modalities and across species.